Olfactory transduction discriminates with great accuracy and sensitivity among a multitude of volatile, low molecular weight compounds (Lancet, D. (1986) Ann. Rev. Neurosci., 9:329-355; Synder, S. H., Sklar, P. B and Prevsner, J. (1988) J. Biol. Chem., 263:13971-13974), but compared to visual transduction, it is relatively poorly understood. One difficulty lies in the heterogeneity of olfactory epithelium in which the olfactory neurons reside. The olfactory primary sensory neurons are located in a pseudostratified columnar epithelium consisting of three principal cells types (Graziadei, P. P. C. (1971) The olfactory mucosa of vertebrates. In: Handbook of Sensory Physiology, Vol. I. (Ed. Beidler, L. M.), Springer-Verlag, Berlin, pp. 27-58). The sustentacular or supportive cells resemble glial cells and stretch from the epithelial surface of the basal lamina. Cell bodies of the sensory neurons lie at various levels in the epithelial layer and extend apical dendrites to the surface of the epithelium and unmyelinated axons through the basal lamina. The third cell type, the basal cell, underlies the receptor neurons and is thought to serve as a precursor population from which new olfactory neurons can arise. Isolation of receptor neurons from these other cell types has been difficult, thereby limiting ability to perform biochemical analysis.
Several attempts have been made to obtain populations of primary olfactory neurons. Initial efforts employed in in vitro culture of the entire olfactory epithelium (Gonzales, et al., (1985) J. Neurosci. Methods, 14:77-90; Nobel et al., (1984) Neurosci. Letts, 45:193-198). N-ethylmaleimide has been used to dissociate olfactory epithelium cells into single cells, which, however, lose excitable properties (Kleene, S. J. and Gesteland, R. C. (1981) Brain Res., 229:536-540). Hirsch, J. D. and Margolis, F. L. (1979) Brain Res., 161:277-291, have employed enzymatic dissociation followed by general mechanical disruption with dissociated cells centrifuged through a bovine serumalbumin (BSA) gradient, yielding a partially purified population of cells. Others (Calof, A. L. and Chikaraishi, D. M., (1989) Neuron, 3:115-127; Pixley, S. K. and Pun, R. Y. K. (1990) Develop. Brain Res., 53:125-130) have devised methods to perform lineage analysis and electrophysiologic studies on embryonic olfactory neuronal cells but the neuronal cells were not in pure cultures and were, therefore, not amenable to biochemical studies.
Thus there is a need in the art for relatively pure populations of primary olfactory neurons which retain their excitability in response to odorants.